Anhydrovinblastine for the treatment of cancer

ABSTRACT

The present invention is particularly directed to the use of a derivative of vinblastine, 3′,4′-dehydrovinblastine (3′,4′-anhydrovinblastine: AHVB), which differs from vinblastine in that it possesses a double bond at the 3′, 4′ position of the catharanthine nucleus rather than the hydroxyl group that is present in the parent structure, in the treatment of cancer.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of copending U.S. patentapplication Ser. No. 09/380,540, filed Mar. 4, 1998, which is a nationalstage of PCT/CA98/00195, filed Mar. 4, 1998. The aforesaid PCTapplication claims priority from Canada Patent Application 2,199,065,filed Mar. 4, 1997; and Canada Patent Application 2,205,314, filed May14, 1997; and Canada Patent Application 2,219,095 filed Oct. 24, 1997.The entire contents of all of the aforementioned applications are herebyincorporated herein by reference.

BACKGROUND OF INVENTION

[0002] The present invention is related generally to the use ofantineoplastic vinca alkaloids as antitumour agents. More particularly,the present invention is related to providing use for a derivative ofvinblastine, anhydrovinblastine (hereinafter AHVB), as an antineoplasticagent with improved therapeutic properties, demonstrating asignificantly higher maximum tolerated dose and less toxicity than itsparent and related compounds.

[0003] Due to a high degree of unpredictability, classic techniques ofdrug development are inventive. Mostly through a process of elimination,a large number of natural products and synthetic chemical compounds arescreened for desired effects, using a series of increasingly complexsystems, beginning with simple in vitro cell-level assays, progressingto animals and finally human clinical trials. But, due to essentialcharacteristics such as adsorption, distribution and metabolism, theinitial in vitro tests that can not take these features into accountcould eliminate a powerful drug that does not perform well in suchsimple systems. The drug could be metabolized to different compounds inanimal models than in humans, which may also demonstrate differentadsorption or distribution patterns. Or finally, compounds can look verypromising all the way through clinical trials, but then demonstrateunpleasant side effects or a high degree of tolerance when used by thehuman population at large. It is never obvious which compound willcontinue to look promising as each stage of tests and development areinitiated.

[0004] Control of tumorous growth has been achieved to a certain degreeusing oncolytic vinca alkaloids as antitumour agents alone or incombination with other antineoplastic drugs in cancer chemotherapy formore than 20 years. Approximately 30 alkaloids with a wide range ofpharmacological activities have been extracted from the Vinca rosea(Catharanthus roseus), commonly known as the periwinkle plant. Of these,only vinleurosine, vinrosidine, vinblastine and vincristine possesssignificant anti-tumour activity. In particular, vinblastine andvincristine have been used widely as single agents and in combinationwith outer antineoplastic drugs in cancer chemotherapy. In addition tothe naturally occurring alkaloids, some vinca alkaloid analogues havebeen synthesized by functional transformation or by semisyntheticprocesses (R. J. Cersosimo, et al., Pharmacotherapy 3:359-274, 1983; P.Mangency, et al., Org. Chem. 44:3765-3768, 1979; R. Maral, et al.,Cancer Lett. 22:49-54, 1984).

[0005] Chemically, these vinca alkaloids have a dimeric asymmetricstructure composed of 2 nuclei linked by a carbon-carbon bond; adihydroindole nucleus (vindoline), which is the major alkaloid containedin the periwinkle, and the indole nucleus catharanthine (FIG. 1). Thestructural difference between vincristine and vinblastine exists at theR1 position while vinblastine and vindesine differ with regard to the R2and R3 substituents.

[0006] The mode of action of the antineoplastic vinca alkaloids has yetto be completely understood. However, it has been established that theantitumour activity is directly related to the high binding affinity ofthese compounds for tubulin, the basic protein subunit of microtubules(R. A. Bender and B. Chabner, In: Chabner (ed) Pharmacol. Princ. ofCancer Treat., Saunders, Phil, Pa., p. 256, 1982; W. A. Creasey, In:Hahn (ed) Antibiotica, Vol. 2, Springer, Berlin, p. 414, 1979). Theconsensus is that these agents arrest cell mitosis at metaphase bypreventing tubulin polymerization to form microtubules and by inducingdepolymerization (R. J. Owellen and C. A. Hartke, Cancer Res.,36:1499-1504, 1976; R. H. Himes and R. N. Kersey, Cancer Res.,36:3798-3806, 1976; R. S. Camplejohn, Cell Tissue Kinet. 13:327-332,1980). As such, the vinca alkaloids are cell cycle-specific anti-mitoticagents, or spindle poisons. The binding affinity of the vinca alkaloidsto tubulin correlates poorly with the relative ability of vincristine,vinblastine and vindesine to inhibit cell growth (R. S. Camplejohn,supra; P. J. Ferguson and C. E. Cass, Cancer Res., 45:5480-5488, 1985).The major difference in anti-tumour activity between these drugsappears, therefore, to relate to their retention in tumour tissue (P.Ferguson, supra; J. K. Horton et al., Biochem. Pharmacol. 37:3995-4000,1988). In a similar vein, the different toxicity profiles of the vincaalkaloids seems related to tissue uptake and retention properties ratherthan to inherent tubulin binding affinity. For example, studies havedemonstrated that vincristine is more potent than vinblastine orvindesine in blocking fast axoplasmic transport in nerve cells (S. Ochsand R. Worth, Proc. Am. Assoc. Cancer Res., 16:70, 1975; S. Y. Chan etal., J. Neurobiol. 11:251-264, 1980). In addition, it is taken up intonerves 4 times faster than the other drugs (Z. Iqbal and S. Ochs, J.Neurobiol., 11:251-264, 1980) and exhibits an extended terminalelimination phase of plasma clearance, suggesting a more prolongedexposure to vincristine than to the other vinca alkaloids (R. L. Nelsonet al., Cancer Treat. Rev., 7:17-24, 1980).

[0007] The in vitro and in vivo differences observed between the vincaalkaloids are striking given the subtle chemical alterations displayedby the various agents relative to their large, complex molecularstructure. For example, vincristine is very effective in treating humanrhabdosarcomas transplanted in nude mice whereas vinblastine is notactive in this system (N. Bruchovsky et al., Cancer Res. 25:1232-1238,1965). This difference is obtained simply as a result of thesubstitution of an aldehyde group for a methyl group at the R1 position.Further, this chemical substitution leads to a shift in the toxicologyprofile such that peripheral neuropathy (in the absence of hematologicaltoxicity) is dose limiting in humans for vincristine whereas anemia andleucopenia are typically dose limiting for vinblastine (W. P. Brads,Proc. Int. Vincaalkaloid Symposium, 95-123, 1980; S. S. Legha, Med.Toxicol., 1:421-427, 1986). A particularly interesting therapeuticprofile has been observed for a new semisynthetic vinca alkaloid named

[0008] Navelbine™ (vinorelbine, 5′-noranhydroblastine). This compound isless potent than vinblastine and vincristine against murine P388 andL1210 leukemia but is active against cells derived from human lungcancer whereas the other vinca alkaloids are inactive (S. Cros, et al.,Seminars in Oncology, 16:15-20, 1989). As well, clinical trials onNavelbine™ support its utility in treating non-small cell lung cancer(A. Depierre et al., Am. J. Clin. Oncol., 14:155-119, 1991; A. Yokoyamaet al., Am. Soc. Clin. Oncol., 11:957, 1992). The toxicity profile ofthis agent appears similar to that of vinblastine, where hematologicaltoxicities and not neurological side effects are dose limiting.

[0009] Vincristine has proved particularly useful as an intravenouslyadministered oncolytic agent in combination with other oncolytic agentsfor the treatment of various cancers including central-nervous-systemleukemia, Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma,rhabdomyosarcoma, neuroblastoma, and Wilma tumor. It is for intravenoususe only and the intratecal administration is uniformly fatal. Followingsingle weekly doses, the most common adverse reaction is hair loss; themost troublesome are neuromuscular in origin. When single weekly dosesof the drug are employed, the adverse reactions of leukopenia, neuriticpain, constipation, and difficulty in walking can occur. Other adversereactions that have been reported are abdominal cramps, ataxia, footdrop, weight loss, optic atrophy with blindness, transient corticalblindness, fever, cranial nerve manifestations, parehesia and numbnessof the digits, polyuria, dysuria, oral ulceration, headache, vomiting,diarrhea, and intestinal necrosis and/or perforation.

[0010] Navelbine™ (vinorelbine tartrate) is a novel vinca alkaloid inwhich the catheranthine unit is the site of structural modification. Itsanti-tumour activity is also thought to be due primarily to its abilityto interfere with microtubule activity thereby inhibiting mitosis atmetaphase through its interaction with tubulin. It is indicated in thetreatment of advanced non-small cell lung cancer as a single agent or incombination, administered by intravenous route only. Its side effectsinclude phlebitia or extravasion injury as it is a moderate vasicant.Studies on adverse reactions based on use of Navelbine™ as a singleagent indicate Granculocytopenia as the major dose-limiting toxicity,although it was generally reversible and not cumulative over time. Mildto moderate peripheral neutopathy manifested by pareathesia andhypesthesia are the most frequently reported neurologic toxicities,occurring in 10% of patients. Mild to moderate nausea occurs in roughlyone-third of patients treated with Navelbine™ with a slightly lesserfraction experiencing constipation, vomiting, diarrhea, anorexia, andstomatitis.

[0011] Compounds exhibiting lessened toxic effects with equal or greaterchemotherapeutic activity remain to be achieved. Thus, a need remainsfor a drug providing improved anti-tumour efficacy for the treatment ofcancer.

[0012] It is, therefore, an object of the present invention to provide amethod of treating cancer which comprises administering to a humanpatient suffering from cancer and in need of treatment, an amount ofAHVB, effective to arrest or significantly slow the progress of thedisease.

[0013] It is another object of the present invention to provide a methodof using AHVB as an antitumour agent, comprising therapeutic amount ofthe chemical substance of the present invention to arrest tumorousgrowth.

[0014] The above and various other objects and advantages of the presentinvention are achieved by administration of a derivative of vinblastine,AHVB. Other objects and advantages will become evident from thefollowing detailed description of the present invention.

BRIEF SUMMARY OF INVENTION

[0015] The present invention is particularly directed to the use of aderivative of vinblastine, 3′,4′-anhydrovinblastine (AHVB), whichdiffers from vinblastine in that it possesses a double bond at the3′,4′position of the caranthine nucleus rather than the hydroxyl groupthat is present in the parent structure, as an antineoplastic agent inthe therapeutic treatment of cancer.

[0016] One embodiment of the present invention involves the use of 3′,4′-anhydrovinblastine, or variants thereof, as an antineoplastic agentin the treatment of cancer.

[0017] Another embodiment of the present invention involves the use of3′,4′-anhydrovinblastine as an antineoplastic agent in the treatment ofcancer, wherein the concentration of 3′,4′-anhydrovinblastine is atsignificantly higher maximum concentration than therapeuticallyacceptable concentrations for vinblastine or Navelbine™ for use in thetreatment of cancer.

[0018] Yet another embodiment of the present invention involves the useof 3′,4′-anhydrovinblastine as an antineoplastic agent in the treatmentof cervical cancer.

[0019] Yet a further embodiment of the present invention involves theuse of 3′,4′-anhydrovinblastine as an antineoplastic agent in thetreatment of lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 depicts the chemical structure of some vinca alkaloids.

[0021]FIG. 2 depicts comparison of effects of administering a singleintraperitoneal injection, at a subacutely toxic dose, of vincristine,Navelbine™ and AHVB to Nb rats bearing single well-developed,subcutaneous Nb2-U17 tumor transplants on average tumor weight andaverage weight of the rat as a function of time.

[0022]FIG. 3 depicts comparison of the effects of administering a singleintraperitoneal injection, at a half subacutely toxic dose ofvincristine, Navelbine™ and AHVB to Nb rats bearing singlewell-developed, subcutaneous Nb2-U17 tumor transplants on average tumorweight and average weight of the rat as a function of time.

[0023]FIG. 4 depicts changes in mean animal wight of BDF1 mice bearingintraperitoneal P388 tumours following i.v. administration of saline,vincristine, Navelbine™ and AHVB.

[0024]FIG. 5 depicts an example cytotoxicity curve used to estimate theIC₅₀ of various vica alkaloids.

[0025]FIG. 6 depicts P388 anti-tumour activity of selected formulationsof vinca alkaloids.

[0026]FIG. 7 depicts a dose response curve obtained for AHVB when usedto treat BDF1 mice bearing P388 tumours.

[0027]FIG. 8 depicts cytotoxicity curves used to estimate the IC₅₀ ofAHVB on the cell lines SKOV3 and C-4.

[0028]FIG. 9 depicts mean tumour weight in grams over time (30 daysperiod) following administration at days 1, 5, and 9, of Navelbine™,bisulphate AHVB, ditartrate AHVB, and control.

DETAILED DESCRIPTION

[0029] There are many possible derivatives or variations of vinblastinepossible. However, there is no certainty, even to those skilled in thearea of anti-cancer drug development, that any such derivatives will beas efficacious or even more efficacious than the parent compound. Thistakes much testing and experimentation.

[0030] The term “variants” for purposes of 3′,4′-anhydrovinblastinemeans any chemical structure that is a derivative of3′,4′-anhydrovinblastine achieved through conservative substitution ofside groups, yet still exhibits the same or similar antineoplasticproperties as 3′,4′-anhydrovinblastine.

[0031] Characterization of AHVB Anti-tumour Activity In Vitro

[0032] Cytotoxicity experiments on AHVB were performed as directcomparisons with vincristine and Navelbine™ in order to assess itsinherent antineoplastic profile against a variety of tumour cell typesrelative to other relevant vinca alkaloids. The cytotoxicity of AHVB wasinvestigated in vitro against a panel of tumour cell lines of varyinglineage in order to determine the specificity of its antitumour activitywith respect to cell type. The tumour lines studied were P388lymphocytic leukemia (a murine lymphocytic leukemia), Noble (Nb) rat U17lymphoma, MCF7 human breast carcinoma, H460 human non-small cell lungcarcinoma, K562 human erythrokeukemia and LS180 human colon carcinomabased on established NCI in vitro new anti-cancer drug cytotoxicityscreening protocols.

[0033] Standard dose response cytotoxicity assays (R. Mosmass, J.Immunol. Meth., 65:55-64, 1983) were utilized to determine the IC50(drug concentration required to induce 50% inhibition of tumour cellgrowth) for vincristine, Navelbine™ and AHVB. The results are presentedin Table 1. The indicated cell lines were obtained from either the ATCCor NCI tumour repository and were cultured in tissue culture media bystandard techniques well known to those skilled in the art, prior todilution to a defined cell concentration required for the studies in 96well plates.

[0034] A wide range of drug concentrations were exposed to tumour cellsgrowing at log phase in 96-well microtitre plates. Cell concentrationsdepended on the cell line as well as the length of time to be cultured.Typically, P388 cells were plated at a concentration of 30,000, 2,000and 750 cells per well for studies lasting 1, 3 and 7 days,respectively. MCF7 cells were plated at a concentration of 7,000 and1,500 cells per well for studies lasting 3 and 7 days, respectively.H460 cells were plated at a concentration of 2,500 and 1,000 cells perwell for studies lasting 3 and 7 days, respectively. K562 cells wereplated at a concentration of 1,500 and 10,000 cells per well for studieslasting 1 and 3 days, respectively. LS180 cells were plated at aconcentration of 5,000 and 20,00 cells per well for studies lasting 3and 7 days, respectively. After plating all cell lines were incubated(CO2 incubator at 37° C., 5% CO2) for 24 hours prior to addition of thecytotoxic agent (See Table 1).

[0035] Subsequently the plates were incubated for the indicated timeperiod. At specified times, cells were washed and subsequently exposedto the dye inclusion marker MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium), whichaccumulated into viable cells. MTT was added to the cells at a finalconcentration of 50 □g per well. After a 4 hour incubation, the cellswere washed free of media and unreacted MTT, prior to addition of DMSOwhich was required to solubilize the insoluble formazan precipitate thatformed in viable cells. After the sample was mixed through repeatedpipetting, the coloured product was measured using a plate readeroperating at 570 nm. The absorbance values obtained for cells culturedin the absence of drug was assumed to represent 100% viability.Experiments were repeated to substantiate any differences noted betweenAHVB and other vinca alkaloids.

[0036] Characterization of AHVB Antitumour Activity In Vivo

[0037] Evaluation of in vitro cell cytotoxicity was followed by studiesregarding the antineoplastic activities of AHVB in three in vivo rodentmodels. Thus, anti-tumour activity of AHVB was determined using a ratsolid tumour model (U17 lymphoma), the murine P388 tumour model (R.Noble, et al., Cancer Res., 37:1455-1460, 1977; P. W. Gout et al.,Biochem Cell Biol., 64:659-666, 1986), and a H460 SC Tumour mouse model.

[0038] The U17 cell line was originally derived from a transplantablemalignant lymphoma that arose spontaneously in male Noble rats (BritishColumbia Cancer Research Centre Joint Animal Breeding Facility withparents obtained from the National Institutes of Health, Bethesda, Md.).The cell line is prolactin dependent and can readily be cultured invitro. U17 derived solid tumours are generated by subcutaneous injection(via the trocar method) of a small (2 mm²) piece of tumour tissueobtained from male Noble rat. Tumour tissue used for the implants arosetwo weeks after injection of 5×10⁶ U17 cells (from culture)subcutaneously in the nape of the neck. For assessing the anti-tumouractivity of AHVB, tumour bearing animals (2-4 gm tumours) were given asingle treatment of drug and tumour size was measured as a function oftime following treatment. The anti-tumour activity was assessed at aseries of different doses in order to determine the maximum therapeuticdose of AHVB. Comparative studies between vincristine, vinblastine andAHVB were performed. For these studies anti-tumour activity wasdetermined at the maximum therapeutic dose of each drug.

[0039] Antitumour studies on mice focussed, in one case, on the P388leukemia model. This is a standard NCI model for evaluation of newanti-cancer agents and it has been demonstrated to be sensitive totreatment with vinca alkaloids. This is an ascitic tumour model that wasgenerated by intraperitoneal inoculation of 1×10⁶ P388 cells (derivedfrom culture, with an original cell line obtained from the NCI tumourrepository) in BDF1 mice (Charles Rivers). One day after tumour cellinoculation, mice were treated with a single intravenous injection ofdrug. Animal weight was monitored daily and tumour progression wasmeasured as an increase in animal weight and through estimation ofsurvival time. Therapy was described by a decrease in tumour progressionand an increase in survival time relative to an untreated control group.Initial studies established the maximum therapeutic dose for AH-VB.Subsequently comparative studies with vincristine and Navelbine™ wereinitiated where animals were treated with each drug at the maximumtherapeutic dose.

[0040] The Canadian Council on Animal Care Guidelines were strictlyadhered to and all animal protocols employed were approved by the AnimalCare Committees of UBC and the BCCA. Animals were evaluated twice dailyfor any signs of stress (tumour or drug related) and if an animalappeared to be suffering (excessive weight loss or gain, lethargy,scruffy coat, etc.) than the animal was terminated.

[0041] Identification of Maximum Tolerated Dose of AHVB

[0042] Range-finding acute (14 day), single dose toxicity studies wereperformed in healthy male Nb rats in order to determine the maximumtolerated dose of vincristine sulfate, Navelbine™ and AHVB whenadministered as a single, intraperitoneal injection in these rodents(see Table 2).

[0043] To this end, healthy non-tumour bearing male Nb rats (weightrange 333-399 grams) were divided in groups of 3 animals. Each group wasused to test one drug at one dosage. In a group, each animal receivedone intraperitoneal injection at a particular dose, as indicated inTable 2. The volumes within which the drugs were administered dependedon the concentration of the drug solution (in saline) and the weight ofthe animals, and ranged from 0.1 -1.0 ml. Saline was used as a control.The highest dose of each drug which allowed survival of all animals in agroup (3 out of 3) was taken as the subacutely toxic dosage for thedrug, i.e. 0.7 mg/kg for vincristine, 2.0 mg/kg for Navelbine™ and 3.0mg/kg for AHVB.

[0044] The health of the animals was assessed by daily weightmeasurements in addition to behavioural indications of stress. Animalscontinued to be monitored throughout the complete 14 day study period.Animals were euthanized in the event of signs of severe stress or weightloss in excess of 20%. All animals were necropsied at the end of thestudy period or at the time of premature euthanasia. Once weight loss inexcess of 20 % or premature animal death was noted at a dose level, thedose was decreased until the weight loss nadir was less than 20% and nopremature animal deaths were observed.

[0045] Studies in the Rat U17 Lymphoma Model

[0046] Cultures of the non-metastatic, pre-T Nb2 lymphoma lineoriginally developed at The University of British Columbia anddesignated Nb2-U17 (Anticancer Research 14:2485-2492, 1994), and areavailable from the British Columbia Cancer Research Centre. Cells fromexponentially growing Nb2-U17 suspension cultures were injectedsubcutaneously into methoxyflurane-anesthetized, mature male Nb rats (5rats; 310 -380 grams; 5×10⁶ cells/rat in 1 m] of culture medium) at thenape of the neck using a 1.5″ 20-gauge needle. At about 3 weeks, whenthe tumours reached a size of 4 -7 cm (length+width), the animals weresacrificed and the tumours used for transplantation as described below.

[0047] A tumour from a rat was excised, minced and the tumour tissue wasput into trocars (2″, 13 gauge). The tissue samples were implantedsubcutaneously in the nape of the neck of methoxyflurane-anesthetizedmale Nb rats (248 -404 grams; 1 trocar per rat). This procedure wasrepeated 5 times to get a total of 60 tumour-bearing rats to be used forefficacy studies of the 3 drugs.

[0048] When the tumours were well established (1.5 -2 weeks later),three separate groups of 20 rats, as closely matched as possible interms of both tumour weight and rat weight, were selected foradministration of the three test articles (i.e. one group for each testarticle).

[0049] Vincristine was administered to rats weighing 281 -384 grams,bearing tumours weighing 6.3 -16.3 grams. Navelbine™ was administered torats weighing 274 -389 grams bearing tumours weighing 9.1 -23.3 grams.AHVB was administered to rats weighing 303 -400 grams, bearing tumoursweighing 7.9 -25.9 grams. Tumour weights were estimated using thehemi-ellipsoid model (weight in grams=length×depth×π/6 in cm).

[0050] The oncolytic effects of each of the three drugs were assessed ata subacutely toxic dose, determined for each drug in preliminary studiesusing non-tumour-bearing, mature male Nb rats, i.e. 3.0, 2.0 and 0.7mg/kg for AHVB, Navelbine™ and vincristine, respectively as illustratedin FIG. 2. In addition, each drug was assessed at 50% and 25% of itssubacutely toxic dose. Five tumour-bearing rats were used to evaluatethe effect at each dose level. The drugs were administeredintraperitoneally as a single bolus in a volume of 0.19 -0.31 ml, asindicated by the weight of the animals. To this end, drug preparationswere diluted to appropriate concentrations using sparged saline adjustedwith acetic acid to pH 4.2. For each drug, a group of 5 control ratsreceived an intraperitoneal injection of the equivalent amount of saline(pH 4.2). The tumour-bearing rats were organized in the followinggroups: Group Drug/Saline Dose (mg/kg) 1 saline — 2 AHVB 3.0 3 AHVB 1.54 AHVB 0.75 5 saline — 6 Navelbine ™ .0 7 Navelbine ™ 1.0 8 Navelbine ™0.5 9 saline — 10 vincristine 0.7 11 vincristine 0.35 12 vincristine0.175

[0051] Following administration of the test articles, the animals;weight and tumour size (using calipers) were determined daily until thetumour reached an estimated weight of 35 grams, or started to ulcerate,at which times the animals were sacrificed (by carbon dioxideinhalation) and subjected to necropsy. Animals were also monitored atleast daily for signs of stress for the full length of the study.Animals manifesting severe symptoms of stress (rapid weight loss,panting, hunched posture, scruffy coat) were also sacrificed and anecropsy performed.

[0052] Anhydrovinblastine Sulfate (3′,4′-dehydrovinblastine) wasobtained from the British Columbia Cancer Agency (BCCA), InvestigationalDrug Section. Vincristine Sulfate (Sulfate of 22-oxovincaleukoblastine)was obtained from David Bull Laboratories Ltd., Australia. Navelbine™(vinorelbine tartrate;3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine-di-L-tartrate) waspurchased from Burroughs Wellcome Inc., Canada; 0.9% Sodium ChlorideInjection USP, pH 4.2 was purchased from Baxter.

[0053] The methodology involving animals was approved by the BCCA'sInstitutional Animal Care Committee (IACC) at UBC prior to conductingthe studies (Animal Care Certificate No. A94-1602). During the study thecare, housing and use of animals was performed in accordance with theCanadian Council on Animal Care Guidelines.

[0054] The results of the efficacy studies are given in FIGS. 2-3. FIGS.2-3 present averages of data from 5 or fewer animals.

[0055] The effect of administering a single intraperitoneal, subacutelytoxic dose of AHVB, Navelbine™ and vincristine on the size of single,well-established Nb2-U17 lymphoma transplants (average weight 10 -13grams) and the weight of the animals, as a function of time aredemonstrated in FIG. 2. Whereas the tumours in the control animalscontinued to increase in size to an average weight of about 40 grams in6 days, the tumours in the drug-treated animals in each case regressedto essential non-palpability within 5 days of drug administration. Afterday 10, recurrence of tumours in Navelbine™-and AHVB-treated animalsoccurred to about the same extent. In contrast, recurrence of tumourswas not observed in vincristine-treated animals (not even on day 29).FIG. 2 also shows that the animals lost weight following drugadministration. However, most of the weight was regained after about 17days. As controls for each drug, Nb2-U17 tumour transplant-bearing ratsinjected with saline were used. For each of the six groups five animalswere used. Vincristine sulfate (0.7 mg/kg) was administered in a volumeof 0.20 -0.23 ml to rats weighing 281 -331 grams bearing tumoursweighing 7.6 -14.2 grams. Navelbine™ (2.0 mg/kg) was administered in avolume of 0.24 -0.31 ml to rats weighing 297 -389 grams bearing tumoursweighing 11.5 -13.7 grams. AHVB (3.0 mg/kg) was administered in a volumeof 0.20-0.24 ml to rats weighing 314 -374 grams bearing tumours weighing8.2 -14.2 grams. Vincristine sulfate controls: saline was administeredin a volume of 0.21 -0.26 ml to rats weighing 294 -370 grams bearingtumours weighing 9.4 -14.6 grams. Navelbine™ controls: saline wasadministered in a volume of 0.25 -0.29 ml to rats weighing 310 -365grams bearing tumours weighing 9.5 -18.2 grams. AHVB controls: salinewas administered in a volume of 0.19 -0.25 ml to rats weighing 303 -400grams bearing tumours weighing 7.9 -16.6 grams. The efficacies of eachdrug were determined separately at three different dosages versus acontrol.

[0056]FIG. 3 shows the anti-tumour effects of the three drugs at 50% oftheir individual maximum tolerated doses. The data show that Navelbine™was less potent than AHVB which in turn was less potent thanvincristine.

[0057] Nb2-U17 tumour transplant-bearing rats injected with saline wereused as controls. For each of the six groups five animals were used.Vincristine sulfate (0.35 mg/kg) was administered in a volume of 0.23-0.27 ml to rats weighing 327 -384 grams bearing tumours weighing 6.4-13.4 grams. NavelbineT (1.0 mg/kg) was administered in a volume of 0.24-0.28 ml to rats weighing 296 -351 grams bearing tumors weighing 9.1-14.1 grams. AHVB (1.5 mg/kg) was administered in a volume of 0.20 -0.23ml to rats weighing 308 -359 grams bearing tumors weighing 9.7 -19.5grams. Vincristine sulfate controls: saline was administered in a volumeof 0.21 -0.26 ml to rats weighing 294 -370 grams bearing tumoursweighing 9.4 -14.6 grams Navelbine™ controls: saline was administered ina volume of 0.25 -0.29 ml to rats weighing 310 -365 grams bearingtumours weighing 9.5 -18.2 grams. AHVB controls: saline was administeredin a volume of 0.19 -0.25 ml to rats weighing 303 -400 grams bearingtumors weighing 7.9 -16.6 grams. The efficacies of each drug weredetermined separately at three different dosages versus a control. InFIG. 3, results of the three drugs at equivalent, i.e. half subacutelytoxic, dosages are compared. The controls in FIG. 3 are the same as inFIG. 2.

[0058] Studies in the Murine P388 Model

[0059] A cytotoxicity curve was generated to estimate the IC₅₀ ofvincristine, Navelbine™ and AHYB in the murine P388 cell line (see FIG.5). In this study, P388 cells derived from an ascitic tumour grown inBDF1 were first separated from red cells employing Ficoll-Paque.Isolated white cells were washed twice then placed in serum containingtissue culture media (1×105 cells per ml of RPMI 1640 supplemented withL-glutamine, penicillin, streptomycin and 10% fetal bovine serum) andcultured for 2 hours. All non adherent cells were collected and thatcell population was defined as P388 cells and used for cytotoxicityassays 24 hours later. Cytotoxicity assays were performed as describedin the the section entitled Characterization of AHVB Anti-tumourActivity In Vitro. The drug concentrations used are indicated on theX-axis. Vincristine is represented by the filled circles, Navelbine™ bythe filled triangles and AHVB by the filled squares.

[0060] The in vivo anti-tumour activity of AHVB was compared to that ofvincristine, Navelbine™ in the BDF1-murine P388 model in the procedureas follows. P388 cells were derived from the ascities of previouslyinjected female BDF1 mice (19 -21 grams) P388 cells, from the NCI tumourrepository were inoculated directly into mice. The cells arrive from NCIfrozen in 1 ml aliquots. These samples were thawed rapidly at 37° C. andsubsequently injected (within 1 hour) intraperitoneally into two mice,0.5 ml per mouse. One week (7 days) after inoculation, the tumour cellswere harvested by removing peritoneal fluid using a sterile syringe witha 22 gauge needle. The cells, pooled from two animals, were countedusing a heamocytometer, diluted (RPMI media) to a concentration of 2×10⁶cells/ml and 0.5 ml was then re-injected into each of two BDF1 mice.Remaining cells were washed and placed into a DMSO containing media andfrozen (in freezer packs that cool at a defined rate). This process wasrepeated weekly over a 2-week period. Cells used for anti-tumour studieswere collected from the third passage to the 20th passage. After the20th passage the cells were no longer used for experimental studies.Newly established cells were derived from the frozen cells prepared asdescribed above.

[0061] Groups (five mice per group) of female BDF1 mice (Charles Rivers,Canada) were injected (intraperitoneal) with 10⁶ P388 cells (asdescribed above). One day after tumor cell inoculation, the mice weregiven a bolus intravenous injection of indicated drug via the lateraltail vein. Control groups were injected with saline. Free drug sampleswere prepared on the day of injection such that the final concentrationswere sufficient to deliver the indicated drug dose in a volume of 200□1. All dilutions were made using 0.9% Sodium Chloride Injection USP.The mice were briefly (less than 30 sec.) restrained during intravenousinjections. Dilation of the vein was achieved by holding the animalsunder a heat lamp for a period of between five and ten minutes.Following administration of the test articles, animals were weigheddaily for fourteen days and monitored for signs of stress twice dailyfor the first 14 days (once daily on weekends) and once daily for theremainder of the study. Severely distressed animals were terminated byCO₂ asphyxiation and the time of death was recorded to occur on thefollowing day. Although complete dose titrations were completed for eachdrug, the data shown in FIG. 6 is that obtained after administration ofthe free drugs at their maximum tolerated dose. This was 3, 40 and 40mg/kg for vincristine, Navelbine™ and AHVB, respectively.

[0062]FIG. 4 presents the results of a study demonstrating vincaalkaloid induced weight loss following a single intravenous injection ofthe indicated drug at the maximum tolerated dose (see FIG. 6). Thesedata were obtained as part of the study detailed in FIG. 6. Aftertreating mice (bearing the P388 tumour) with a single dose of theindicated drug, animals were examined twice daily for the first 14 days(once daily on weekends). Mean body weight was determined daily overthis time period and the results are shown in FIG. 4. Weight gain in thecontrol is an indication of tumour progression. Results indicate thatAHVB, administered at 40 mg/kg, is the least toxic of the three drugsevaluated.

[0063] The dose response curve obtained for AHVB when used to treat BDF1mice bearing P388 tumours is presented in FIG. 7. The studies wereconducted as described for FIG. 6. The maximum tolerated dose of AHVB(40 mg/kg) as specified in these studies reflects a very acute (within 1hour) toxic reaction that limits further dose excalatin for i.v.administration of AHVB. This contrasts the more prolonged toxicityobserved for Navelbine™ at its maximum tolerated dose and suggests thatan ability to circumvent the acute toxicity of AHVB could lead tosignificant increases in its maximum tolerated dose.

[0064] Based on observation of the in vitro drug screen studies, it issurprising that AHVB would perform well as an antineoplastic agent foruse in cancer therapy. The in vitro tests indicate that AHVB isconsistently 10 to 15 fold less active on per molar basis (Table 1 andFIG. 5) than vincristine and Navelbine™. These results suggest that AHVBwould not perform well as an anti-tumour agent. However, in an efficacystudy, also employing the P388 cell line (see FIG. 6), the anti-tumouractivity of AHVB at the maximum tolerated dose (40 mg/kg, single i.v.injection) is significantly better than that observed for vincristine(administered at the maximum tolerated dose of the free drug of 3mg/kg). Improved anti-tumour activity, in this case, is measured by thenumber of long term survivors (>60 days). It is important to stressthat, for this example, AHVB is approximately 10 times less toxic (on aweight basis) than vincristine. Therefore, 10 times more drug can begiven and it is at this dose that improvements were observed in the longterm survival of animals with P388 tumours. When compared to Navelbine™,the in vivo results are even more surprising as the maximum tolerateddose of the two drugs in animals bearing P388 tumours are about the same(40 mg/kg).

[0065]FIG. 8 shows the cytotoxicity of AVHB on SKOV3 cells and C-4 cellswith a 3 day incubation. The IC₅₀s for the SKOV3 and C-4 cells were 4.0□M and 0.02 □M respectively. Both cell lines were obtained from the ATCCand grown using standard growth techniques and medium as describedabove. The IC₅₀s were determined through standard cytoxicity assaysdescribed above, with each well containing approximately 10⁴ cells.

[0066] Studies in the H460 SC Tumour Mouse Model

[0067] Cultures of H460 Human Lung cells are available from the BritishColumbia Cancer Research Center. Cells were injected subcutaneouslytwice into mature male Rag-2 mice (24 mice, 1×10⁶ cells/mouse) using a26-gauge needle. The H460 cells were suspended in a Hank=s Balanced SaltSolution without calcium. Tumours were allowed to form in the mice for11 days.

[0068] When the tumours were well established, four separate groups ofmice, were selected for administration of the three test articles (i.e.one group for each test article of AHVB bisulphate, AHVB ditartrate, andNavelbine™) and one control.

[0069] AHVB bisulphate and ditartrate, and Navelbine™ were solubilizedusing 5% dextrose saturated with Argon. Both of these articles were at aconcentration of 20 mg/ml. Any dose dilutions were made with 5%dextrose.

[0070] The articles were administered intravenously on the days 1, 5 and9, as were controls of 5% dextrose. Body weights and tumour measurementswith calipers were taken every day for the first 10 days and then everyother day for the remainder of the study.

[0071] Following administration of the test articles, the animals;weight and tumour size (using calipers) were determined daily for thefirst 10 days and then every other day for the remainder of the study.If the tumour size reached 1 gram in weight or the tumour started toulcerate, the animals were sacrificed (by carbon dioxide inhalation) andsubjected to necropsy. Animals were also monitored at least daily forsigns of stress for the full length of the study. Animals manifestingsevere symptoms of stress (rapid weight loss, panting, hunched posture,scruffy coat) were also sacrificed and a necropsy performed.

[0072] Anhydrovinblastine Sulfate (3′,4′-dehydrovinblastine) wasobtained from the British Columbia Cancer Agency (BCCA), InvestigationalDrug Section. Navelbine™ (vinorelbine tartrate;3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine-di-L-tartrate) waspurchased from Glaxo/Burroughs Wellcome Inc., Canada.

[0073] The methodology involving animals was approved by the BCCA'sInstitutional Animal Care Committee (IACC) at UBC prior to conductingthe studies (Animal Care Certificate No. A94-1602). During the study thecare, housing and use of animals was performed in accordance with theCanadian Council on Animal Care Guidelines.

[0074] The results of the efficacy studies are given in FIG. 9 andpresent averages of data from 6 or fewer animals. Each mouse in a givenarticle group had two subcutaneous tumours on its back. Each tumour wasmeasured in length and width and the volume of each tumour wascalculated by (L X W)²/2. The two tumour volumes were then averaged. Thevolume averages of all the mice/group were averaged to yield a mean forthe single date point appears on the graph in FIG. 9. The calculationwas performed each day the tumours were measured. The standard deviationof the mean and the standard error of the mean were calculated with theerror bars appearing in the graph in FIG. 9.

[0075] Studies in the C-4 (Cervical) Solid Tumour Model

[0076] Cultures of C-4 Human Cervical Carcinoma cells are available fromthe British Columbia Cancer Research Centre. Cells were injectedsubcutaneously twice into mature male Rag-2 mice (24 mice, 1×10⁶cells/mouse) using a 26-gauge needle. The C-4 cells were suspended in aHank=s Balanced Salt Solution without calcium. Tumours were allowed toform in the mice for 31 days.

[0077] When the tumours were well established, four separate groups ofmice, were selected for administration of the three test articles (i.e.one group for each test article of AHVB bisulphate, AHVB ditartrate, andNavelbine™) and one control.

[0078] AHVB bisulphate and ditartrate, and Navelbine™ were solubilizedusing 5% dextrose saturated with Argon. These articles were administeredat doses of 20 mg/Kg I.V. Any dose dilutions were made with 5% dextrose.

[0079] The articles were administered intravenously on the days 1, 5 and9, as were controls of 5% dextrose. Body weights and tumour measurementswith calipers were taken regularly over the period of the study of 69days.

[0080] Following administration of the test articles, the animals;weight and tumour size (using calipers) were determined regulary overthe period of the study. If the tumour size reached 1 gram in weight orthe tumour started to ulcerate, the animals were sacrificed (by carbondioxide inhalation) and subjected to necropsy. Animals were alsomonitored at least daily for signs of stress for the full length of thestudy. Animals manifesting severe symptoms of stress (rapid weight loss,panting, hunched posture, scruffy coat) were also sacrificed and anecropsy performed.

[0081] Anhydrovinblastine Sulfate (3′,4′-dehydrovinblastine) wasobtained from the British Columbia Cancer Agency (BCCA), InvestigationalDrug Section. Navelbine™ (vinorelbine tartrate;3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine-di-L-tartrate) waspurchased from Glaxo/Burroughs Wellcome Inc., Canada.

[0082] The methodology involving animals was approved by the BCCA'sInstitutional Animal Care Committee (IACC) at UBC prior to conductingthe studies (Animal Care Certificate No. A94-1602). During the study thecare, housing and use of animals was performed in accordance with theCanadian Council on Animal Care Guidelines.

[0083] The results of the efficacy studies are given in Table 3 andpresent averages of data from 6 or fewer animals. Each mouse in a givenarticle group had two subcutaneous tumours on its back. Each tumour wasmeasured in length and width and the volume of each tumour wascalculated by (L X W)²/2. The two tumour volumes were then averaged. Thevolume averages of all the mice/group were averaged to yield a mean foreach single date point. The calculation was performed each day thetumours were measured.

[0084] Navelbine™ tumours reached their observable >growth threshold=atday 41 and continued to grow steadily whereas the AHVB ditartratereached the threshold on day 55. The tumour treated with AHVB bisulphateshowed negligible tumour growth through day 69. Navelbine™ had an 84%delay in growth in the tumour, AHVB ditartrate had an extended delay of106%, and AHVB bisulphate exhibited a marked delay in tumour growth ofgreater than 209%. Tumour growth did not reach the observable growththreshold over 70 days. This data is found in Table 3.

[0085] Taken together, the results presented here show that AHVB hassignificant and unique pharmacological properties in vivo that lead tosignificant improvements in in vivo antitumor efficacy relative to othervinca alkaloids such as vincristine and Navelbine™. These results areunique and new in that the in vivo activity of AHVB predicted it to besignificantly less on the basis of in vitro cytotoxicity studies.

[0086] The present invention also provides pharmaceutical compositionscontaining a compounds as disclosed in the claims in combination withone or more pharmaceutically acceptable, inert or physiologicallyactive, diluents or adjuvants. The compounds of the invention can befreeze dried and, if desired, combined with other pharmaceuticallyacceptable excipients to prepare formulations for administration. Thesecompositions may be presented in any form appropriate for theadministration route envisaged. The parenteral and the intravenous routeare the preferential routes for administration.

[0087] 3′,4′-anhydrovinblastine may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques. In addition, there isprovided a pharmaceutical formulation comprising3′,4′-anhydrovinblastine and a pharmaceutically acceptable carrier.3′,4′-anhydrovinblastine may be present in association with one or morenon-toxic pharmaceutically acceptable carriers and/or diluents and/oradjuvants and if desired other active ingredients. The pharmaceuticalcompositions containing 3′,4′-anhydrovinblastine may be in a formsuitable for oral use, for example, as tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsionhard or soft capsules, or syrups or elixirs.

[0088] Compositions intended of oral use may be prepared according toany known to the art for the manufacture of pharmaceutical compositionsand such compositions may contain one or more agents selected from thegroup consisting of sweetening agents, flavouring agents, colouringagents and preserving agents in order to provide pharmaceuticallyelegant and palatable preparations. Tablets contain the activeingredient in admixture with non-toxic pharmaceutically acceptableexcipients which are suitable for the manufacture of tablets. Theseexcipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate: granulating and disintegrating agents for example, cornstarch, or alginic acid: binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

[0089] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

[0090] Aqueous suspensions contain active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethyene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more colouringagents, one or more flavouring agents or one or more sweetening agents,such as sucrose or saccharin.

[0091] Oily suspensions may be formulated by suspending the activeingredients in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions may contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents such as thoseset forth above, and flavoring agents may be added to provide palatableoral preparations. These compositions may be preserved by the additionof an anti-oxidant such as ascorbic acid.

[0092] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavouring andcolouring agents, may also be present.

[0093] Pharmaceutical compositions of the invention may also be in theform of oil-in-water emulsions. The oils phase may be a vegetable oil,for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monoleate, and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monoleate. The emulsions may also contain sweetening andflavouring agents.

[0094] Syrups and elixirs may be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative and flavouringand colouring agents. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. thissuspension may be formulation according to known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also besterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

[0095] 3′,4′-anhydrovinblastine may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

[0096] 3′,4′-anhydrovinblastine may be administered parenterally insterile medium. The drug, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

[0097] For the compounds of this invention, the dose to be administered,whether a single dose, multiple dose, or a daily dose, will vary withthe particular compound being used. Factors to consider when decidingupon a dose regimen include potency of the compound, route ofadministration, size of the recipient and the nature of the patient'scondition.

[0098] The dosage to be administered is not subject to defined limits,but in will usually be an effective amount. It will usually be theequivalent, on a molar basis of the pharmacologically active free formproduced from a dosage formulation upon the metabolic release of theactive free drug to achieve its desired pharmacological andphysiological effects.

[0099] An oncologist skilled in the art of cancer treatment will be ableto ascertain, without undue experimentations, appropriate protocols foreffective administration of the compounds of this present invention byreferring to the earlier studies of vinblastine and its derivatives.

[0100] AHVB a derivative of the Vinca Alkaloid Vinblastine has shownsignificant cytotoxic potential against a panel of human cancer celllines, and significant activity against the human H460 non-small celllung carcinoma tumour xenograph in SCID/Rag-2 Mice. In vitrocytotoxicity assays utilizing the MTT cytotoxicity assay with a drugexposure time of 72 hours have shown that AHVB is an active cytotoxicdrug with IC₅₀ values ranging from 20-24 nM against the H460 humannon-small cell lung carcinoma, C-4 human cervical carcinoma, K562 humanleukemia, and the A431 human epidermoid cell lines. AHVB wasapproximately 10-fold less active than NavelbineT when tested in vitroagainst the same cell lines. Surprisingly, however when AHVB was testedin vitro in solid tumour efficacy experiments it was found to be morepotent than Navelbine™. Male SCID/Rag-2 mice were inoculated sc. withH460 cells and after 12 days of tumour growth AHVB and Navelbine™ weredelivered i.v. at doses of 10mg/kg and 20 mg/kg on days 1, 5, 9. In thismodel, AHVB caused greater tumour growth inhibition and was less toxicthan Navelbine™. These results suggest that AHVB may have desirablepharmacological properties for therapeutic applications.

[0101] It is to be understood that the examples described above are notmeant to limit the scope of the present invention. It is expected thatnumerous variants will be obvious to the person skilled in the art towhich the present invention pertains, without any departure from thespirit of the present invention. The appended claims, properlyconstrued, form the only limitation upon the scope of the presentinvention. TABLE 1 Relative Cytotoxicity Of Vincristine, Ahvb AndNavelbine ™ On Tumor Cell Lines EXPO- SURE DRUG IC₅₀ (nM) CELL TIME VIN-NAVEL- LINES TYPE (days) CRISTINE BINE AHVB P388 murine 1 11.0

 3.6 20.0

 10.0 140.0

 53.0 leukem- 3  1.0

 0.3 0.7

 0.3 15.0

 8.7 ia 7 2.0 2.5 20.0 MCF7 human 1 N.D. N.D. N.D. breast3 >2500 >2500 >2500 7  2.6

 1.6 2.6

 1.6  31.3

 12.4 H460 human 1 N.D. N.D. N.D. lung 3 3.5 0.3 10.0 7 2.5 >0.5 5.0K562 human 1 >50.0 >50.0 >50.0 erythro- 3  1.5

 0.4 2.5

 2.2 18.8

 8.8 leukem- 7 N.D. N.D. N.D. ia LS180 human 1 N.D. N.D. N.D. colon3 >50.0 >50.0 >50.0 7 1.5 0.5 17.5

[0102] TABLE 2 Estimation of subacutely toxic dosages of vincristinesulfate, Navelbine ™, and AHVB when administered to healthy male Nb ratsas a single, intraperitoneal injection. Dose Drug (mg/kg) Mortality(surviving rats/injected rats) 1 ml Saline pH 4.3 n/a 3/3 Vincristinesulfate 1.0 0/3 0.7 3/3 0.6 3/3 0.5 3/3 Navelbine ™ 10.0 0/3(Vinorelbine 5.0 0/3 tartrate) 3.0 2/3 2.0 3/3 1.0 3/3Anhydrovinblastine 10.0 0/3 5.0 2/3 4.4 0/1 4.0 ½ 3.0 3/3

[0103] TABLE 3 Solid Tumour Delay in Growth Data DOSE 20 mg/kg INITIALGROWTH % DELAY IN EXPERI- IV days (day) GROWTH MENT 1, 5, 9 TOTAL OFEXPT. (DIG) C-4effl Control (Saline) 32 2 Navelbine ™ 59 29 84 AHVBBisulphate 99 69 209 AHVB Ditartrate 66 36 106

What is claimed is:
 1. The use of 3′,4′-anhydrovinblastine, or variantsthereof, as an anti-neoplastic agent in the treatment of cancer.
 2. Theuse of 3′,4′-anhydrovinblastine as an antineoplastic agent in thetreatment of cancer, wherein the concentration of3′,4′-anhydrovinblastine is at significantly higher maximumconcentration than therapeutically acceptable concentrations forvinblastine or Navelbine ™ for use in the treatment of cancer.
 3. Atherapeutic composition comprising 3′,4′anhydrovinblastine and one ormore pharmaceutically acceptable, inert or physiologically activediluents or adjuvants.
 4. A therapeutic composition comprising3′,4′anhydrovinblastine and one or more pharmaceutically acceptable,inert or physiologically active diluents or adjuvants.
 5. The use of thecomposition of claims 3 or 4 for the treatment of cancer.
 6. The use ofthe composition of claims 3 or 4 for the treatment of cervical cancer.7. The use of the composition of claims 3 or 4 for the treatment of lungcancer.
 8. The use of 3′,4′anhydrovinblastine for the manufacture of amedicament for the treatment of cancer.
 9. The use of the composition ofclaims 3 or 4 for the manufacture of a medicament for the treatment ofcancer.